Istituto di Biofisica, Consiglio Nazionale delle Ricerche (CNR), Via De Marini, 6, 16149, Genoa, Italy.
Cell Mol Life Sci. 2013 Mar;70(5):923-33. doi: 10.1007/s00018-012-1172-5. Epub 2012 Oct 4.
The cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is an anion channel activated by protein kinase A phosphorylation. The regulatory domain (RD) of CFTR has multiple phosphorylation sites, and is responsible for channel activation. This domain is intrinsically disordered, rendering the structural analysis a difficult task, as high-resolution techniques are barely applicable. In this work, we obtained a biophysical characterization of the native and phosphorylated RD in solution by employing complementary structural methods. The native RD has a gyration radius of 3.25 nm, and a maximum molecular dimension of 11.4 nm, larger than expected for a globular protein of the same molecular mass. Phosphorylation causes compaction of the structure, yielding a significant reduction of the gyration radius, to 2.92 nm, and on the maximum molecular dimension to 10.2 nm. Using an ensemble optimization method, we were able to generate a low-resolution, three-dimensional model of the native and the phosphorylated RD based on small-angle X-ray scattering data. We have obtained the first experiment-based model of the CFTR regulatory domain, which will be useful to understand the molecular mechanisms of normal and pathological CFTR functioning.
囊性纤维化跨膜电导调节因子（CFTR），囊性纤维化的缺陷蛋白，是一种受蛋白激酶 A 磷酸化激活的阴离子通道。CFTR 的调节域（RD）有多个磷酸化位点，负责通道的激活。该结构域本质上是无规则的，使得结构分析成为一项艰巨的任务，因为高分辨率技术几乎无法应用。在这项工作中，我们通过互补的结构方法获得了天然和磷酸化的 RD 在溶液中的生物物理特性。天然 RD 的回旋半径为 3.25nm，最大分子尺寸为 11.4nm，大于相同分子量的球状蛋白的预期尺寸。磷酸化导致结构的紧缩，回旋半径显著减小至 2.92nm，最大分子尺寸减小至 10.2nm。我们使用集合优化方法，根据小角度 X 射线散射数据生成了天然和磷酸化 RD 的低分辨率三维模型。我们已经获得了 CFTR 调节域的第一个基于实验的模型，这将有助于理解正常和病理 CFTR 功能的分子机制。
